Ultrasensitive and On-field Detection of a Plant Virus by a Nanotube-filtering Device and Isothermal Amplification

Commodities

Vegetables:
tomatoes

Practices

Crop Production: plant breeding and genetics

Education and Training: decision support system, mentoring

Pest Management: disease vectors, prevention

Proposal abstract:

An isothermal amplification of nucleic acids coupled with a top-notch nanotube filter that increases detection of plant viruses in field conditions is proposed. Spotted wilt of tomato, a devastating virus disease that can lead up to 100% of losses, will be used as case study. By detecting the disease in early stages and without the need of expensive equipment or laboratory services for diagnosis, farmers and extension agents will be able to plan better management strategies for insect vectors and weeds that act as reservoirs of viruses. The system proposed couples two stages. First is a Recombinase Polymerase Amplification, a sensitive technique that produces millions of copies of a DNA or RNA target to detectable levels by naked eye or portable instruments, at a constant temperature close to body temperature. In conjunction, the second stage is a micro device that traps viruses in a nanotube forest, eliminating host contaminants and detection inhibitors, as well as increasing limits of detection by concentrating all the viruses present in a sample, according to their size. The complete system is expected to be portable, affordable and with enough sensitivity and specificity to be deployed for on-site detection of plant viruses. This project aims to increase yield and profit for farmers, as well as to alleviate environmental hardship by decreasing the number of insecticide sprays used to control insects that spread plant viruses.

Project objectives from proposal:

Hypothesis: A recombinase Polymerase Amplification (RPA) can be engineered for on-field detection of Tomato spotted wilt virus with great sensitivity and specificity. Additionally the use of the 3D nanotube-based filter CNT-STEM will allow an increased detection and reliability for the detection technique. By providing our field-friendly virus-capture device and combining it with RPA that doesn’t need bulky and non-portable laboratory equipment, we expect to provide farmers and field pathologists with a system that allows early on-field virus detection, has a great sensitivity and does not require professional training or shipment to a laboratory.

Specific objectives are:

1. To develop and validate a RPA reaction for detection of Tomato spotted wilt virus

2. To integrate CNT-STEM as a virus capture platform with the molecular detection of genomic fragments of RPA in asymptomatic infected tomato seedlings or just-virus-inoculated tomato plants

3. To compare the detection of TSWV in RPA-only and RPA+CNT-STEM for field samples

Any opinions, findings, conclusions, or recommendations expressed in this publication are
those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.

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